The present invention relates to novel polynucleotides, and to the use of these polynucleotides, for insertional mutagenesis and gene tagging in fungi. The invention also relates to collections of fungus mutants obtained by random insertion of the Impala transposon from Fusarium oxysporum into the genome of these fungi. These collections of mutants represent an effective genetic tool for studying the function of genes in fungi.
Transposons may be defined as mobile genetic elements capable of moving between two DNA sequences. By virtue of their capacity to insert into genes (exons, introns, regulatory regions), they can be the cause of mutations. Because of this, they contribute to the evolution of the genomes in which they exist as a parasite. Transposons have been classified in two groups depending on their propagation method (Finnegan, 1989; Capy et al., 1998):
Class I elements (retroelements) transpose via an RNA intermediate which is reverse-transcribed into DNA by a reverse transcriptase. This class is subdivided into retroelements or retrotransposons which may or may not be bordered by LTRs (long terminal repeats). Among the LTR retroelements are the elements of the gypsy family and of the copia family. They have genes which are homologous to the gag and pol genes of retroviruses and differ in the organization of the various functional domains of the pol gene. In addition, the gypsy family has a gene which is homologous to env which, in retroviruses, contributes to their infectiousness. Among the non-LTR retroelements LINEs which have gag and pol genes and a poly-A sequence are distinguished, and also SINEs, which also have a poly-A tail but lack gag and pol sequences, are distinguished; they are presumed to derive from prior LINE elements (Eickbush, 1992; Okada and Hamada, 1997);
Class II elements transpose via a mechanism of excision and reinsertion of the transposon DNA sequence. Their general structure consists of two inverted repeat sequences (ITRs) bordering an open reading frame encoding a transposase required for the transposition of the element. These elements have been grouped together into superfamilies, according to the sequence homologies of their transposases and/or of the ITRs, including that of the Tc1/mariner elements (Doak et al., 1994), of the Fot1/Pogo elements (Capy et al., 1998; Smit and Riggs, 1996), of the hAT elements (Calvi et al., 1991), of the P elements (Kidwell, 1994) or of the CACTA elements (Gierl 1996).
The identification and study of fungus transposons is of very great value, in particular with a view to developing tools for insertional mutagenesis (Brown et al., Curr. Opin. Microbiol. 1:390-4, 1998) and also for studying the genome of these fungi (Dobinson et al., Trends in Microbiology, 1:348-3652, 1993).
Various strategies have therefore been implemented for identifying transposons in the genome of fungi. The first and second take advantage of the knowledge which derives from previously characterized elements. This involves the use of heterologous probes used in Southern hybridization experiments or amplifications using oligonucleotides derived from highly conserved domains, such as that of the LTR retroelement reverse transcriptase. The third strategy consists in characterizing repeat DNA sequences. In this case, differential hybridization between the genomic DNA and a ribosomal probe is required. Transposons of the Fot1 family have thus been identified in the Magnaporthe grisea genome (Kachroo et al., Current Genetics 31:361-369, 1997; Farman et al., Mol. Gen. Genetics 251:675-681 1996; Kachroo et al., Mol. Gen. Genetics 245:339-348, 1994). The final method, unlike the previous three, makes it possible to identify functional and active elements; this is the transposon trap. This strategy uses the inactivation of a marker gene in which the mutation engendered by the insertion of the element can be identified using a positive screen. Thus, the am (glutamate dehydrogenase) gene has made it possible to characterize the Tad retroelement, which is of the LINE type, in Neurospora crassa, following its reinsertion into this gene (Kinsey and Helber, 1989). The niaD (nitrate reductase) gene of Aspergillus nidulans has also been used for trapping transposons. Specifically, a mutation which inactivates this gene confers chlorate resistance. Various transposons have thus been identified in Fusarium oxysporum (Daboussi et al., Genetica 93:49-59, 1994) and in Aspergillus (U.S. Pat. No. 5,985,570). The class II element Fot1 from Fusarium oxysporum was the first transposon identified using inactivation of the niaD gene (Daboussi et al., 1992). In addition, the use of the niaD gene in Fusarium oxysporum has made it possible to trap the Impala transposon belonging to the superfamily of the Tc1/mariner-type elements (Langin et al., 1995). Various Impala transposon subfamilies have been identified in Fusarium oxysporum (Hua-Van et al., Mol. Gen. Genetics 259:354-362, 1998). The transposition of the Impala element has been studied in Fusarium oxysporum. When the Impala transposon is integrated into the promoter or the introns of a given gene, it may then inactivate the expression of this gene. On the other hand, after transposition of the Impala transposon, the gene is reactivated, which constitutes a positive control for the transposition event. Such a strategy for identifying the transposition has been used in Fusarium with a construct comprising the Impala transposon integrated into the promoter regulatory sequence of the nitrate reductase (niaD) gene from Aspergillus nidulans (Hua-Van, 1998).
It has not been possible to demonstrate the transposition of Impala, other than at an extremely low rate which is incompatible with the development of a tool for insertional mutagenesis, using the niaD/Impala gene construct of the pNi160 plasmid (Langin et al., 1995) in other fungi, and more particularly Magnaporthe grisea. These observations suggest that the niaD/Impala construct of the pNi160 plasmid (Langin et al., 1995), and more particularly that the Impala transposon itself, are not functional in other fungi, and in particular in M. grisea. 
Now, such a tool for creating a collection of insertion mutants in the fungus genome, and more particularly the genome of pathogenic filamentous fungi, is essential for studying their genome and for studying the function of their genes. Analyzing the functions of fungus genes is essential for discovering novel antifungal compounds which can be used for treating fungal conditions in human or animal health or for agriculture.
The present invention relates to novel polynucleotides comprising a marker gene which is functional in Magnaporthe grisea and which is inactivated by the insertion of an Impala transposon. These polynucleotides make it possible to demonstrate the transposition of the Impala element in fungi with a transposition rate which is compatible with the development of tools for insertional mutagenesis. A subject of the invention is also therefore methods for preparing fungus mutants by inserting an Impala transposon into their genome and methods for identifying a fungus gene associated with a phenotype of interest. Finally, the invention relates to collections of fungus insertion mutants and uses thereof.
Polynucleotides
The present invention therefore relates to a polynucleotide, in particular an isolated or purified polynucleotide, comprising a marker gene which is inactivated by the insertion of an Impala transposon, such that said marker gene comprises, in the direction of transcription, a promoter regulatory sequence which is functional in Magnaporthe grisea and which is functionally linked to the coding sequence of said marker gene.
The transformation of a fungus with a polynucleotide according to the invention and the excision of the transposon lead to the expression of the marker gene. The detection of the marker gene expression thus makes it possible to monitor the transposition events and to select the insertion mutants. The selection of the mutants can be improved by labeling the transposon with a second marker gene which is different from the first. This second marker gene makes it possible to monitor the reinsertion of the transposon into the genome of the fungus. A subject of the invention is also therefore polynucleotides as described above, in which the Impala transposon comprises a marker gene.
The marker genes used are suitable for selecting the transposition events by means of a simple screen. Any marker gene, the expression of which in a fungus can be detected with a phenotypic screen, may be used in the present invention. It is understood that the term xe2x80x9cmarker genexe2x80x9d also denotes chimeric genes comprising genetic elements of different origins. The marker genes of the present invention may thus combine a promoter sequence from a fungus and a coding sequence of a marker gene which is not from a fungus.
According to the invention, the expression xe2x80x9cpromoter regulatory sequence which is functional in Magnaporthe griseaxe2x80x9d is intended to mean any polynucleotide which allows the expression, in Magnaporthe grisea, of a coding sequence to which it is functionally linked. The techniques which make it possible to determine whether a promoter sequence is functional in Magnaporthe grisea are known to those skilled in the art. For example, Magnaporthe may be transformed with a polynucleotide comprising, in the direction of transcription, a potential promoter sequence and a reporter gene. Monitoring the expression of the reporter gene in the fungus transformed with the polynucleotide makes it possible to determine whether this promoter sequence is functional in Magnaporthe. Any promoter sequence can thus be tested in order to determine its functionality in Magnaporthe grisea. The promoter regulatory sequence may be a promoter regulatory sequence of a gene from Magnaporthe grisea, or may originate from another fungus, and more particularly from another filamentous fungus. Advantageously, the promoter regulatory sequence which is functional in Magnaporthe grisea consists of the promoter regulatory sequence of a fungal nia (nitrate reductase) or gpd gene. Preferably, the promoter regulatory sequence which is functional in Magnaporthe grisea consists of a promoter regulatory sequence, which is functional in Magnaporthe, of the niaD (Malardier et al., 1989) or gpda gene from Aspergillus nidulans (Punt et al., 1990). In order to be functional in Magnaporthe, the promoter regulatory sequence of the niaD gene from Aspergillus nidulans is preferably longer than 337 bp, than 0.4 kb, than 0.5 kb, than 0.6 kb, than 0.7 kb, than 0.8 kb, than 0.9 kb, and more preferably longer than or equal to approximately 1 kb. In a preferred embodiment of the invention, the promoter sequence which is functional in Magnaporthe grisea consists of a 1.3 kb polynucleotide corresponding to the intergenic fragment between the niaD and niiA genes from Aspergillus nidulans (Genbank M58291; Johnstone et al., Gene 90:181-192, 1990; Punt et al., 1995). It is understood that a partial functional regulatory sequence, which is not functional per se, but the random integration of which into the genome of Magnaporthe grisea downstream of a Magnaporthe grisea promoter allows the expression of the marker gene, is not a regulatory sequence which is functional in Magnaporthe grisea according to the present invention.
According to a preferential embodiment of the invention, the coding sequence of the marker gene is chosen from the coding sequences of a reporter gene, the expression of which is easily measured, in particular GUS (U.S. Pat. No. 5,268,463, U.S. Pat. No. 5,599,670) or GFP (U.S. Pat. No. 5,491,084, U.S. Pat. No. 5,741,668), the coding sequences for a gene for tolerance to an antibiotic or a herbicide, such as the genes for resistance to hygromycin (hph: Punt et al., 1987), to phleomycin (ble: Drocourt, 1990) or to the herbicide bialaphos (Bar: Pall and Brunelli, 1993), or a gene for resistance to sulfonylureas (Sweigard et al., 1997). According to another preferential embodiment of the invention, the marker gene is chosen from the sequences of genes encoding enzymes, which are functional in fungi. Advantageously, the marker gene is the nitrate reductase gene. When the fragment according to the invention is integrated into an niaxe2x88x92 fungus, the strain transformed with this construct conserves a mutant phenotype. The appearance of nia+ colonies on a minimum medium (NaNO3 as the only nitrogen source) reveals the excision of the Impala transposon allowing the expression of the niaD gene. These nia+ revertants can be selected on this medium due to their dense and aerial phenotype which is different from the low flat phenotype of the niaxe2x88x92 colonies. The use of the niaD gene as a marker requires the use of an niaxe2x88x92 fungus. The methods for identifying niaxe2x88x92 fungi are well known to those skilled in the art. Mention will in particular be made of the method described by Daboussi et al. (1989).
The polynucleotides of the present invention comprise a marker gene as described above which is inactivated by the insertion of an Impala transposon (Langin et al., 1995; Hua-Van et al., 1998). The Impala transposon comprises an open reading frame encoding the functional transposase bordered by inverted repeat sequences (ITRs). It inserts at a TA dinucleotide, which becomes duplicated. The excision of Impala is imprecise and most commonly leaves a three-nucleotide footprint corresponding to the left or right end of the element, in addition to the TA dinucleotide duplicated during the insertion. The point of insertion of the Impala transposon into the marker gene must therefore be chosen such that, following excision, the residual footprint does not prevent the expression of the marker gene. Preferably, the Impala transposon is inserted into the promoter sequence or into an intron of the marker gene. Several copies of Impala have been identified in Fusarium oxysporum and comparing their sequences has made it possible to define three subfamilies having ITRs of variable length and sequence (Hua-Van et al., 1998). In a preferred embodiment, the polynucleotides of the present invention comprise an Impala 160 transposon. The Impala 160 element comprises 1 280 bp, and it is bordered by two inverted repeat sequences of 27 bp framing an open reading frame encoding a 340 amino acid transposase (Langin et al., 1995; Genbank S75106). In a preferred embodiment, the polynucleotides of the present invention comprise the 1.3 kb promoter of the niaD gene from Aspergillus nidulans, functionally linked to the coding sequence of the niaD gene from Aspergillus nidulans, and an Impala 160 transposon inserted into the promoter of the niaD gene. In a particularly advantageous embodiment of the invention, the polynucleotides of the present invention comprise the pNiL160 plasmid. These constructs are used to transform an niaxe2x88x92 fungus and the insertion mutants resulting from the transposition of the Impala element are selected for their nia+ phenotype on a minimum medium. In another preferred embodiment, the polynucleotides of the present invention comprise the promoter of the gpd gene from Aspergillus nidulans, functionally linked to the coding sequence of the hph gene for resistance to hygromycin, and an Impala 160 transposon inserted into the promoter of the gpd gene. These polynucleotides are used to transform a fungus and to select the hygromycin-resistant insertion mutants resulting from the transposition of the Impala element.
Any Impala transposon may be used in the constructs and the methods of the present invention. It is understood that the term xe2x80x9cImpala transposonxe2x80x9d also denotes modified Impala transposons. Among these modifications mention will be made in particular of the insertion of a marker gene or of activator sequences into the Impala transposon, or the inactivation of the transposase in order to obtain a defective Impala transposon. The construction of these modified transposons uses conventional molecular biology techniques which are well known to those skilled in the art.
The polynucleotides of the present invention are preferentially used to obtain insertion mutants of fungi. Inserting the Impala transposon into a gene generally leads to the total or partial inactivation of this gene. The use of an Impala transposon comprising activator sequences makes it possible, on the other hand, to obtain overexpression mutants. The transposon modifications thus allow the use of various methods of insertional mutagenesis (Bancroft et al., Mol. Gen. Genet. 233:449-461, 1992; Bancroft and Dean, Mol. Gen. Genet. 240:65-67, 1993; Long et al., PNAS 90:10370-10374, 1993).
The present invention therefore also relates to a polynucleotide as described above, comprising an Impala transposon into which a marker gene is inserted between the two ITRs of the transposable element without affecting the functionality of the transposase, thus making it possible to have an autonomous and labeled element. All the marker genes, the use of which is envisioned for observing the excision of the Impala transposon, may also be used for labeling said transposon in a preferred embodiment of the invention. Preferably, the marker gene is inserted downstream of the sequence encoding the transposase and upstream of the left ITR (at the NheI site). The insertion of a marker gene into the Impala transposon allows better selection of the insertion mutants. Alternatively, a truncated marker gene may be inserted into the Impala transposon. The use of a marker gene lacking a promoter makes it possible to use the polynucleotides of the present invention as a promoter trap. The use of a marker gene comprising a truncated promoter makes it possible to use the polynucleotides of the present invention as a trap for activator sequences.
Finally, the present invention relates to a polynucleotide as described above, comprising a defective Impala transposon, i.e. a transposon in which the transposase of the Impala element has been inactivated, in particular by mutation, by deletion, by insertion of a marker gene or by replacement with a marker gene. The transposition of this defective Impala element is more easily controlled in the insertional mutagenesis methods of the present invention. The construction of a defective Impala element in which the transposase is inactivated uses conventional molecular biology techniques which are known to those skilled in the art (Sambrook et al., 1989). In one embodiment of the invention, the open reading frame encoding the transposase of the Impala element is replaced with a marker gene expressed under the control of a promoter which is functional in Magnaporthe grisea. The coding sequence of the transposase may, for example, be replaced with the gene for resistance to hygromycin, the gene for resistance to bialaphos or the GFP gene, expressed under the control of a heterologous promoter which is functional in fungi. The defective Impala transposon conserves these insertion sequences (ITRs) and the transposition thereof may therefore be activated in trans, using a transposase placed, for example, on a replicative plasmid.
The polynucleotides of the present invention are preferably inserted into a vector. This vector can be used for transforming a host organism, such as a bacterium for example, and for replicating the polynucleotides of the present invention in this host organism. Preferably, the polynucleotides of the present invention are inserted into a vector for transforming fungi. These vectors are used for replicating or for integrating these polynucleotides into the genome of fungi. Vectors which allow the replication and the integration of polynucleotides into a host organism are well known to those skilled in the art.
Insertional Mutagenesis and Genetic Tagging
The present invention also relates to the use of the polynucleotides described above, for preparing insertion mutants of fungi and for studying the genome of these fungi.
A subject of the present invention is therefore also a method for preparing insertion mutants of fungi, comprising the following steps:
said fungus is transformed with a polynucleotide as claimed in the invention comprising a marker gene which has been inactivated by the insertion of an Impala transposon, under conditions which allow the excision of the Impala transposon of said marker gene and its reinsertion into the genome of the fungus;
the insertion mutants expressing the marker gene are identified.
It is understood that, in the methods according to the invention, the Impala transposon may be modified, and in particular modified by the insertion of a marker gene or of activation sequences. In a preferred embodiment, the Impala transposon comprises a marker gene and the insertion mutants expressing the two marker genes are selected.
Any fungus may be transformed with a polynucleotide according to the invention in order to prepare insertion mutants of this fungus. Mention will be made in particular of the ascomycetes, basidiomycetes and oomycetes. Preferably, the invention relates to the fungi of the Alternaria, Aspergillus, Botrytis, Cladosporium, Claviceps, Colletotrichum, Erysiphe, Fusarium, Mycosphaerella, Phytophthora, Pseudocercosporella, Pyrenophora, Rhynchosporium, Sclerotinia, Stagonospora, Venturia and Ustilago genera. Mention will also be made of the fungi of the Gaeumannomyces, Helminthosporium, Puccinia and Rhizoctonia genera. Preferentially, the invention relates to the fungi of the Magnaporthe and Penicillium genera. More preferentially, the invention relates to the fungi of the Aspergillus fumigatus, Aspergillus nidulans, Botrytis cinerea, Erysiphe graminis, Mycosphaerella graminicola, Penicillium funiculosum and Stagonospora nodorum species. Even more preferentially, the invention relates to Magnaporthe grisea. 
The techniques for transforming fungi are well known to those skilled in the art. Mention will be made in particular of the transformation of protoplasts using PEG, electroporation, transformation with Agrobacterium (De Groot et al., Nature Biotechnology 16:839-842, 1998) or the methods of bombardment using a particle gun (Chaure et al., Nature Biotechnology 18:205-207, 2000).
The transformants are then screened for the expression of the marker gene in order to identify or to select the insertion mutants resulting from the transposition of the Impala element. The marker gene of the polynucleotides of the present invention makes it possible to identify or select insertion mutants by means of a phenotypic screen. By way of example, this screen may be resistance to an antibiotic, resistance to a chemical compound or the measurement of the level of expression of a reporter gene. Various marker genes are described in greater detail above. When the niaD gene is used as the marker gene in an niaxe2x88x92 fungus, the insertion mutants are selected by virtue of their dense and aerial appearance on minimum medium containing NaNO3 as the only nitrogen source.
In order to analyze the insertion mutants thus obtained, it may be advantageous to stabilize the transposon so as to avoid any new transposition. This control of new reinsertion of the transposon may be disregarded if the mutants are tested at a complement close to or below the rate of transposition of the transposition element. In order to control the excision of the transposon, a two-component system may be prepared (Hua-Van, 1998; Kempken and Kuck, 1998). The latter involves the construction of a defective Impala element in which the transposase is inactivated, in particular by mutation, by deletion or by replacement with a marker gene. In this case, the defective Impala transposon is mobilized using a transposase, the expression of which is tightly controlled, thus making it possible to stabilize the Impala element.
A subject of the present invention is therefore also a method for preparing insertion mutants of fungi, characterized in that it comprises the following steps:
said fungus is transformed with a polynucleotide comprising a marker gene which has been inactivated by the insertion of a defective Impala transposon as claimed in the invention;
the defective Impala transposon is mobilized using a transposase, the expression of which is controlled, under conditions which allow the excision of the defective Impala transposon, its reinsertion and its stabilization in the genome of the fungus;
the insertion mutants expressing the marker gene are identified.
The methods which make it possible to control the expression of a gene, such as the Impala element transposase gene, in fungi are well known to those skilled in the art. In a particular embodiment, the fungus is transformed with two polynucleotides; the first polynucleotide comprises the defective Impala transposon, while the second polynucleotide comprises the coding sequence of the Impala element transposase under the control of its own promoter or of a heterologous promoter. The coding sequence of the transposase may be placed on a replicative plasmid or on an integrative plasmid. In order to control the expression of the transposase, the latter may be placed under the control of an inducible promoter. The induction of the expression of the transposase allows the transposition of the defective Impala element and the preparation of insertion mutants, and then the transposon is stabilized when the transposase is no longer expressed. Any inducible promoter which is functional in fungi may be used in the methods of the present invention. Use may in particular be made of the promoter of the nitrate reductase gene from Magnaporthe grisea; specifically, this promoter allows the expression of the nia gene on a minimum medium in the presence of nitrate as the only nitrogen source, whereas the expression of this gene is totally suppressed in the presence of ammonium (Lau and Hammer, 1996). Alternatively, the transposase is, for example, placed on a replicative plasmid carrying a selection marker, this plasmid not being maintained when there is no selection pressure. In this case, the transposase may be expressed under the control of a constitutive promoter or of its own promoter. In the presence of a selection pressure, the maintaining of the replicative plasmid allows the expression of the transposase which, in turn, allows the transposition of the Impala element and the production of insertion mutants. In the absence of selection pressure allowing the replicative plasmid to be maintained, the transposase is lost and the transposon is stabilized in the mutants. The means necessary for preparing such a plasmid are well known to those skilled in the art. By way of example, the transposase may be placed in the pFAC1 replicative vector containing telomeric ends from Podospora (Barreau et al., 1998).
Insertional mutagenesis is a very effective tool for identifying novel genes of interest and for studying their function. In a preferred embodiment, a collection of insertion mutants is screened for a phenotype of interest. Any detectable phenotype may be sought in the insertion mutants of the present invention. Mention will be made in particular of phenotypes relating to the biology, physiology, development and biochemistry of fungi. Preferably, insertion mutants of pathogenic fungi are prepared and the phenotypes sought in these mutants relate to the pathogenicity of these fungi. The phenotypic screen may be based on direct observation of the fungus, on an enzymatic activity measurement, on measuring sensitivity to a fungicide or on studying the virulence of the fungus. When an insertion mutant with a phenotype of interest has been identified, the gene into which, or close to which, the Impala transposon has inserted is isolated. The gene of interest thus tagged by the insertion of the Impala element is isolated using molecular biology techniques which are well known to those skilled in the art. Among the techniques used, mention will be made in particular of the amplification techniques which allow the amplification of a polynucleotide when only the sequence of one end of the polynucleotide is known (in this case, the sequence of the transposon integrated into the genome). These techniques comprise, in particular, inverse PCR (Ochmann et al., Genetics, 120:621-623, 1988; Williams, Biotechniques 7: 762-769, 1989), vectorette PCR (Arnold and Hodgson, PCR Methods Appl. 1:39-42, 1991) and panhandle PCR (Jones and Winistorfer, PCR Methods Appl. 2:197-203, 1993). These techniques make it possible to amplify, to clone and to sequence the sequences flanking the Impala transposon in the genome of the fungus. These flanking sequences are then used to isolate the entire gene inactivated by the insertion of the transposon.
The present invention therefore also relates to a method for identifying a gene associated with a detectable phenotype in fungi, characterized in that it comprises the following steps:
insertion mutants are prepared by inserting an Impala transposon into the genome of said fungi according to one of the methods described above;
at least one insertion mutant with this detectable phenotype is selected;
the gene into which, or close to which, the Impala transposon has inserted is isolated.
Host Organisms
The present invention also relates to a host organism transformed with a polynucleotide of the present invention. According to the invention, the term xe2x80x9chost organismxe2x80x9d is in particular intended to mean any monocellular organism or multicellular organism, which may be a lower or higher organism, in particular chosen from bacteria and fungi. Advantageously, the bacteria are chosen from Escherichia coli. In a preferred embodiment, the invention relates to a fungus transformed with a polynucleotide of the present invention. Preferably, the fungus is chosen from ascomycetes, basidiomycetes and oomycetes. Preferentially, the fungi are chosen from the fungi of the Alternaria, Aspergillus, Botrytis, Cladosporium, Claviceps, Colletotrichum, Erysiphe, Fusarium, Mycosphaerella, Phytophthora, Pseudocercosporella, Pyrenophora, Rhynchosporium, Sclerotinia, Stagonospora, Venturia and Ustilago genera. Mention will also be made of the fungi of the Gaeumannomyces, Helminthosporium, Puccinia and Rhizoctonia genera. Preferentially, the fungi are chosen from Magnaporthe and Penicillium. Advantageously, the fungi are chosen from the Aspergillus fumigatus, Aspergillus nidulans, Botrytis cinerea, Erysiphe graminis, Mycosphaerella graminicola, Penicillium funiculosum and Stagonospora nodorum species. In a particularly advantageous manner, the host organism is Magnaporthe grisea. 
The polynucleotide may be integrated into the genome of the fungus or placed on a replicative plasmid. The present invention therefore also relates to a fungus into the genome of which is integrated a polynucleotide according to the invention. The present invention also relates to insertion mutants of filamentous fungi chosen from the fungi of the Magnaporthe or Penicillium genera, into the genome of which is integrated the Impala transposon.
The reinsertion of Impala into the genome of the fungus makes it possible to generate a collection of insertion mutants of this fungus. The mutants thus obtained may be used for studying the genome of filamentous fungi.
The examples hereinafter make it possible to illustrate the present invention without, however, seeking to limit the scope thereof. All the methods or operations described below in these examples are given by way of examples and correspond to a choice, made from the various methods available to achieve the same result. This choice has no bearing on the quality of the result and, consequently, any suitable method may be used by those skilled in the art to achieve the same result. Most of the methods for engineering the DNA fragments are described in xe2x80x9cCurrent Protocols in Molecular Biologyxe2x80x9d Volumes 1 and 2, Ausubel F. M. et al. or in Sambrook et al., 1989.